product information and testing for depositor material

Product Information and Testing for Depositor Material

©2013 WiCell Research Institute The material provided under this certificate has been subjected to the tests specified and the results and data described herein are accurate based on WiCell's reasonable knowledge and belief. Appropriate Biosafety Level practices and universal precautions should always be used with this material. For clarity, the foregoing is governed solely by WiCell’s Terms and Conditions of Service, which can be found at http://www.wicell.org/privacyandterms.

Product Information

Testing Performed by WiCell

Test Description Test Provider Test Method Test Specification Result

Post-Thaw Viable Cell Recovery WiCell SOP-CH-305 Recoverable attachment after passage

Pass

Identity by STR UW Molecular Diagnostics Laboratory

PowerPlex 16 HS System by Promega

Defines profile Pass

Sterility Biotest Laboratories ST/07 Negative Pass Mycoplasma WiCell SOP-QU-004 Negative Pass Karyotype by G-banding WiCell SOP-CH-003 Report karyotype Pass

Testing Performed by Depositor

No testing performed by Depositor.

Date of Lot Release Quality Assurance Approval

04-April-2013

10/11/2016

X AMKAMKQuality AssuranceSigned by: Klade, Anjelica

Product Name LT2e-H9CAGGFP WiCell Lot Number DB0001 Depositor Life Technologies Banked by Life Technologies Thaw Recommendation Thaw 1 vial into 2 wells of a 6 well plate. Culture Platform Feeder Dependent

Medium: hES Medium

Matrix: MEF

Protocol Because this material was produced by the cell line depositor, WiCell recommends using the depositor protocol included in the CoA and testing results packet.

Passage Number p68

These cells were cultured for 68 passages prior to freeze. The Jump-In R4 clone (C23) was derived from WA09 at p50 and the iCAGG clone was derived from C23 at p60. Add +1 to the passage number to best represent the overall passage number of the cells at thaw.

Date Vialed 06-April-2012

Vial Label LT2e-H9CAGGFP P8 06-April-2012

Biosafety and Use Information Appropriate biosafety precautions should be followed when working with these cells. The end user is responsible for ensuring that the cells are handled and stored in an appropriate manner. WiCell is not responsible for damages or injuries that may result from the use of these cells. Cells distributed by WiCell are intended for research purposes only and are not intended for use in humans.

FORM SOP-QU-004.01

Version B

Edition 01

Assay performed and reported by: MWReviewed by: JB

Equipment ID: 539 Berthold

A1 A2 B1 B2

1 LT2e-H9CAGGFP JB#10702 148 142 145 82 74 78 0.54 Negative

2 Positive (+) Control 218 213 215.5 10470 10437 10453.5 48.51 Positive

3 Negative (-) Control 347 351 349 39 38 38.5 0.11 Negative

Mycoplasma Report

RP/CDM-LRT #10702

Testing Performed by WiCell

Sample Number and ID Comments/Suggestions

A

Average

B

Average

Reading A Reading B

Mycoplasma Results

Ratio

B/A

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

1 2 3

Ra

tio

B/A

Sample Number

Mycoplasma Test RP/CDM-LRT # 10702

Date of Sample:Passage#:Cell Line:

Specimen:

Cell Line Gender:

Reason for Testing:

Investigator:

Total Analyzed:

Results:

Cell:

Karyotype

Total Counted:

Slide Type:

Band Resolution:Total Karyotyped:

Interpretation:

Completed by:

Slide:

Reviewed and Interpreted by:

4

This is a normal karyotype. No clonal abnormalities were detected at the stated band level of resolution.

46,XX

20

LT2e-H9CAGGFP 10702

8

400 - 450

12

Female

2/6/2013

Lot release testing

Chromosome Analysis Report: 009894

hESC

29

Wednesday, February 13,2013

A signed copy of this report is available upon request.

Limitations: This assay allows for microscopic visualization of numerical and structural chromosome abnormalities. The size of structural abnormality that can be detectedis >3-10Mb, dependent upon the G-band resolution obtained from this specimen. For the purposes of this report, band level is defined as the number of G-bands perhaploid genome. It is documented here as “band level”, i.e., the range of bands determined from the four karyograms in this assay. Detection of heterogeneity of clonalcell populations in this specimen (i.e.,mosaicism) is limited by the number of metaphase cells examined, documented here as “# of cells counted”.

This assay was conducted solely for listed investigator/institution. The results may not be relied upon by any other party without the prior written consent of the Director ofthe WiCell Cytogenetics Laboratory. The results of this assay are for research use only. If the results of this assay are to be used for any other purpose, contact theDirector of the WiCell Cytogenetics Laboratory.

Date:_______________________

Date Reported:

Sent To:_____________________Sent By:____ QC Review By: ____

CDM

CG(ASCP), PhD, FACMG

Page 1 of 1 WiCell Research Institute - WiCell Cytogenetics Lab - PO Box 7365 Madison - Wisconsin - 53707-7365

of 6 Human PSC feeder-free culture protocol

Life Technologies Corporation• 7335 Executive way • Frederick, MD 21704 • Phone: 800 955 6288 • www.lifetechnologies.com

Version A 13 March 2012

Reagents and special tools:

1. StemPro® hESC SFM kit (Life Technologies#A1000701, which contains 500 ml DMEM/F12 with Glutamax, 10

ml STEMPRO® hESC Supplement, 50 ml 25% Bovine Serum Albumin )

2. Basic fibroblast growth factor (Life Technologies#PHG0026)

3. -Mercaptoethanol (55 mM, Life Technologies#21985-023)

4. GeltrexTM (Life Technologies#12760-021)

5. Dispase (Life Technologies#17105-041)

6. DPBS without CaCl2 and MgCl2 (Life Technologies#14190-250)

7. Dimethyl sulfoxide (DMSO, Sigma-Aldrich#D2650)

8. StemPro EZPassge disposal stem cell passaging tool (Life Technologies#23181-010)

9. Cell scraper (BD Falcon#353085)

10. Knockout serum replacer (KSR, Life Technologies#10828-028)

Solution and medium preparation

Basic FGF Solution (10 g/ml, For 1 ml)

Basic FGF 10 g

PBS 996 l

25% BSA 4 l

Aliquot and store at -20oC for up to 6 months

Dispase Solution (1 mg/ml, For 50 ml)

Dispase 50 mg

DMEM/F12 50 ml

Sterilize through 0.22 μm filter and store at 4oC for up to 7 days.

StemPro hESC Medium (For 100ml)

DMEM-F12 90.8 ml

STEMPRO® hESC Supplement 2 ml

25% BSA 7.2 ml

-Mercaptoethanol 182 μl

Basic FGF solution 80 μl

Add bFGF (final concentration 8 ng/ml) and -Mercaptoethanol (final concentration 0.1 mM) at the time of medium

change. Medium lasts for up to 7 days at 4oC.




StemPro cryo-preservation medium A

StemPro hESC Medium (50%) + KSR (50%)

StemPro cryo-preservation medium B

StemPro hESC Medium (80%) + DMSO (20%)

Make fresh cryo-preservation medium A and B.

Coating with GeltrexTM

1. Thaw GeltrexTM at 2 to 8°C overnight.

2. Remove DMEM/F-12 from 2 to 8°C storage. Dilute Geltrex TM with DMEM/F-12 (1:30) and mix gently.

3. Cover the whole surface of each culture plate and dishes with the GeltrexTM solution (1 ml for each well of 6-well plate,

1.5 ml for a 60-mm dish, and 3-4 ml for a 100 mm dish).

4. Seal each dish with parafilm to prevent from drying up, and incubate 1 hour at 37°C or overnight at 4°C.

5. If the coated plates or dishes are not used right away, store the coated plates or dishes at 2 to 8°C for up to 1 week.

Transfer plates or dishes to a laminar flow hood and allow them to equilibrate to room temperate (about 1

hour) before use.

Thawing and plating human PSCs

1. Coat culture dishes with Geltrex at least 1 hour before thawing human PSCs. Wear eye protection as cryo-vials

stored in the liquid phase of liquid nitrogen may accidentally explode when warmed.

Note: One 60 mm coated dish for one vial of frozen human PSCs (frozen vial contains cells from ½ of 60mm

dish).

2. Wear ultra low temperature cryo-gloves. Remove a cryo-vial of PSCs from the liquid nitrogen storage tank using

metal forceps.

3. Roll the vial between your gloved hands until the outside is free of frost. This should take between 10-15 seconds.

4. Immerse the vial in a 37oC water bath without submerging the cap. Swirl the vial gently.

5. When only an ice crystal remains, remove the vial from the water bath.

6. Quickly remove the sticker or copy the information written on the vial in your notebook. The writing may come

off the vial after spraying outside of vial with 70% ethanol.

7. Spray outside of the vial with 70% ethanol and place it in hood

8. Pipette cells gently into a sterile 15 ml conical tube using a 1ml pipette.

9. Add 1 ml pre-warmed StemPro hESC medium into the vial to collect resident cells.




11. Using a pipette to remove StemPro hESC medium from the vial and add it to the 15 ml conical tube drop-wise.

While adding the medium, gently move the tube back and forth to mix the human PSCs. This reduces osmotic shock

to the human PSCs.

12. Centrifuge PSCs at 200 x g for 5 minutes and aspirate the supernatant.

13. Re-suspend the cell pellet in 5 ml StemPro hESC medium.

14. Centrifuge PSCs at 200 x g for 5 minutes and aspirate the supernatant.

15. Re-suspend the cell pellet in 5 ml StemPro hESC medium.

16. Aspirate Geltrex solution and label the Geltrex coated 60 mm dish with the passage number from the vial, the

date and your initials.

17. Slowly add the human PSC suspension into the dish.

18. Place the dish gently into the incubator and move the dish in several quick back-and-forth and side-to-side

motions to disperse cells across the surface of the dish.

19. Replace spent medium daily. If feeding more than one dish, use a different pipette for each dish to reduce risk of

contamination. Examine cells under a microscope and colonies may be very small in the first 2 days.

20. Observe PSCs every day and passage cells whenever the colonies are too big or crowded. The ratio of splitting

depends on the total number of PSC colonies in culture plate or dish (Approximately 1:3 for human PSCs at the

first time of recovery).

Passaging human PSCs

Note: This protocol is for the culture on culture dishes and is not for human PSCs cultured in flasks because it is

difficult to remove differentiated colonies in a flask. Also, StemPro EZPassge disposal stem cell passaging tool

and cell scraper cannot reach cells in flask.

1. Coat culture dishes with Geltrex at least 1 hour before passage. Warm appropriate amount of dispase

solution and StemPro hESC medium to 37°C in a water bath.

2. Remove PSC plates and dishes from incubator. Label differentiated colonies with a microscope marker.

3. Aspirate the spent medium with a Pasteur pipette. Remove differentiated colonies with a Pasteur pipette by

aspirating.

4. Gently add 1 ml pre-warmed dispase solution to each well of 6-well plate, 3 ml to each 60 mm dish or 4 ml to

each 100 mm dish.

5. Incubate for 3-5 minutes at 37oC until the edges of cell colonies begin to curl off.

6. Aspirate dispase solution and rinse with DPBS two times gently. Add 1 ml pre-warmed StemPro hESC SFM

to each well of 6-well plate, 2 ml to each 60 mm dish or 3 ml to each 100 mm dish.

7. Roll StemPro EZPassge disposal stem cell passaging tool across the entire dish or plate in one direction

(left to right). When rolling, overlap the next area with the area rolled previously. Rotate the culture




dish or plate 90 degree, and roll StemPro EZPassge disposal stem cell passaging tool across the entire dish

or plate. When rolling, overlap the next area with the area rolled previously. This procedure produces

relatively uniform size of cell clumps.

8. Use a cell scraper to gently detach the cells off the surface of the plates and dishes.

9. Gently transfer cell clumps using a 5-mL pipette and place into a 15 or 50 ml conical tube.

Note: Do not break cell clumps into small pieces.

10. Add 1 ml pre-warmed StemPro hESC SFM to each well of 6-well plate, 2 ml to each 60 mm dish or 3 ml to

each 100 mm dish to collect residual cells and add cell suspension to the tube.

11. Aspirate Geltrex solution from Geltrex coated vessels. Add 2.5 ml pre-warmed StemPro hESC SFM into each

well of 6-well plate, 5 ml into each 60 mm dish or 10 ml into each 100 mm dish.

12. Gently shake the tube to distribute cell clumps evenly. Add appropriate amount of PSC suspension into

each well of culture plate or dish according to split ratio.

Note: The split ratio is variable, though generally between 1:4 and 1:6. Occasionally cells will grow at a

different rate and the split ratio will need to be adjusted. A general rule is to observe the last split ratio and

adjust the ratio according to the appearance of the PSC colonies. If the cells look healthy and colonies have

enough space, split using the same ratio, if they are overly dense and crowding, increase the ratio, and if the cells

are sparse, decrease the ratio. Cells will need to be split every 4-6 days based upon appearance.

12. Place vessels gently in an incubator and move culture vessels in several quick back-and-forth and side-to-

side motions to disperse cells across the surface of vessels.

13. Gently change media the next day to remove non-attached cells, and change StemPro hESC SFM every day

thereafter.

14. Observe cells every day and passage cells whenever the colonies are too big or crowded (approximately every

3 to 4 days).

Cryo-preserving human PSCs cultured in StemPro hESC SFM

1. Warm appropriate amount of dispase and StemPro hESC medium to 37°C in a water bath.

2. Aspirate the medium and gently add 1 ml of dispase solution into each well of 6-well plate, 2 ml

into a 60 mm dish and 4 ml into a 100 mm dish.

3. Incubate for 3-5 minutes at 37°C until the edges of cell colonies begin to curl off.

4. Aspirate dispase solution and rinse with DPBS two times gently, and then add 1 ml StemPro hESC medium

into each well of 6-well plate, 2 ml into a 60 mm dish and 4 ml into a 100 mm dish.











8. Wash vessels with 3 ml of StemPro hESC medium to collect resident cells and add cell suspension to

the tube.

9. Centrifuge at 200 x g for 5 minutes.

10. Gently aspirate supernatant and re-suspend the cells with 1 ml StemPro cryo-preservation medium A for all

cells from one 60 mm dish.

11. Add same volume of StemPro cryo-preservation medium B drop-wise.

12. Allocate 1 ml cell suspension into each cryotube and freeze cells at -80 °C overnight in isopropanol

chamber.

13. Transfer cells into liquid nitrogen tank next day for long term storage.

Change of PSCs cultured on mouse embryonic fibroblasts (MEFs) to StemPro hESC

SFM

If human PSCs are maintained on MEFs, use the following protocol to switch PSCs to feeder-free condition.

1. Coat culture dishes with Geltrex at least 1 hour before experiment.

2. When PSCs cultured on MEFs in dish or plate reach 80-90% confluence, aspirate spent medium and

rinse cells with PBS two times.

3. Add 1 ml StemPro hESC medium into each well of 6-well plate, 2 ml into a 60 mm dish and 4 ml

into a 100 mm dish.








7. Wash vessels with 3 ml of StemPro hESC medium to collect resident cells and add cell suspension to

the tube.

8. Aspirate Geltrex solution from Geltrex coated vessels. Add 2.5 ml pre-warmed StemPro hESC SFM into each

well of 6-well plate, 5 ml into each 60 mm dish or 10 ml into each 100 mm dish.




9. Gently shake the tube to distribute cell clumps evenly. Add appropriate amount of PSC suspension into

each well of culture plate or dish according to split ratio (generally 1:3 to 1:4 ).

10. Place vessels gently in an incubator and move culture vessels in several quick back-and-forth and side-to-

side motions to disperse cells across the surface of vessels.

11. Gently change media the next day to remove non-attached cells, and change StemPro hESC SFM every day

thereafter.

12. Observe cells every day and passage cells whenever the colonies are too big or crowded (approximately every

3 to 4 days).

Note: The contaminated MEFs will die off after 2-3 passages in StemPro hESC SFM.

product information and testing for depositor material

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